Optical flow cell assembly incorporating a replaceable transparent flow cell

ABSTRACT

A new liquid flow cell assembly for light scattering measurements is disclosed which utilized a floating manifold system. The assembly operates with minimal stacked tolerances by aligning the cell to the windows within a manifold and independently aligning the cell to the read head directly. This configuration enables the ability to replace the flow cell or the flow cell/manifold assembly within a light scattering instrument without the need to realign the flow through elements with the light scattering illumination source while still maintaining reproducible, quality data. Some embodiments employ wide bore cells which enable the measurement of process analytic technology (PAT) including online monitoring of reactions.

PRIORITY

This application is a continuation of U.S. patent application Ser. No.15/727,398, filed Oct. 6, 2017.

Related Applications and Patents

The following patents relate to the measurement of light scattering froma liquid sample contained in an optical flow-through cell and are herebyincorporated by reference:

-   U.S. Pat. No. 4,616,927, S. D. Phillips, J. M. Reece, and P. J.    Wyatt, “Sample cell for light scattering measurements,” issued Oct.    14, 1986.-   U.S. Pat. No. 4,907,884, P. J. Wyatt and S. D. Phillips, “Sample    cell monitoring system,” issued Mar. 13, 1990.-   U.S. Pat. No. 5,404,217, G. R. Janik and J. F. Magolske, “Laser    liquid flow cell manifold system and method for assembly,” issued    Apr. 4, 1995.-   U.S. Pat. No. 6,452,672 B1, S. P. Trainoff, “Self cleaning optical    flow cell,” issued Sep. 17, 2002.-   U.S. Pat. No. 7,982,875 B2, S. P. Trainoff, “Method and apparatus    for measuring the scattered light signals from a liquid sample,”    issued Jul. 19, 2011.

BACKGROUND

Throughout this specification, the term “particle” refers to theconstituents of liquid sample aliquots that may be molecules of varyingtypes and sizes, nanoparticles, virus like particles, liposomes,emulsions, bacteria, colloids, etc. Their size range may lie between lnmand several thousand micrometers.

Solutions containing solutes such as molecules, viruses, nanoparticles,liposomes, etc. are often analyzed after their constituent fractions areseparated by liquid chromatography technique such as size exclusionchromatography (SEC), which is also referred to as high performanceliquid chromatography (HPLC) or another separation technique such asfield flow fractionation (FFF), hydrophobic interaction chromatography(HIC), or ion exchange chromatography (IEX). Such measurements mayinclude determination of solute concentration, solution viscosity, andlight scattering properties. The latter measurement used in combinationwith a corresponding concentration determination may be used to derivethe size, molar mass, aggregation, and associations of the solutionsconstituent elements. To improve these measurements the light scatteringdetection is frequently performed by measuring the light scattered bythe separated sample at a plurality of angles with respect to thedirection illuminating light beam. This technique of measuring theintensity of the light scattered by a liquid sample as a function ofangle is referred to as multiangle light scattering (MALS).

MALS measurements may also be performed in a “batch mode” wherein anunfractionated, prepared liquid sample contained within a scintillationvial or cuvette is placed into the path of the illuminating beam. Analternative to the traditional batch measurement wherein the sample isinjected, unfractionated into a flow cell is generally referred to as“stop-flow” or “microbatch” mode. In microbatch mode, after ameasurement is made, the sample is removed from the flow cell by aninjection of another sample or solvent through the flow cell inlet. Thepresent invention is equally relevant to both microbatch and thestandard flow-through measurements discussed above.

While flow through MALS cells have taken many forms through the years,the ease and reliability of MALS measurements took a dramatic stepforward with the introduction of an axial flow cell described byPhillips, et. al. in U.S. Pat. No. 4,616,927 (issued Oct. 14, 2986). Thebasic structure of the axial cell assembly as described by Phillips isshown in FIG. 1. A right circular glass cylinder 101 contains a smallpolished bore 102 drilled through a diameter about midway between thecylinder's base and top. Flow through fixtures 103 and 108 contain achannel 104 through which a liquid sample may pass. These fixtures alsohouse optical windows 105 which are held into the fixtures by retainingelements 106. A seal is maintained between the window 105 and thechannel 104 by a gasket or o-ring. Fluid passes from the inlet fixture103 through a connection tube 107 that directs the sample to flowthrough the cylindrical flow cell 101 and then through the exit fixture108. From there the sample may flow to waste or another detector or asample reclamation system. A light beam 109, generally from a lasersource, is directed to pass through the optical windows, 105 along thesame path as the liquid sample. This entire assembly 100 was then placedinto a read head with spaces milled therein rigidly hold the elements inplace as well as possible. In general, a plurality of photodetectors(not shown) are also rigidly held within the read head positionedcircumferentially about the center of the flow cell. Thesephotodetectors gather light scattered from the light beam by the sampleas it passes through the bore 102. Once the flow cell assembly wasfitted into the read head, the laser was aligned such that the beam 109passes through the center of the bore 102 without grazing its walls.

A problem associated with all flow through optical cells, and inparticular those which measure static light scattering, is theinevitable presence of contaminants accumulating within the cell itself.These contaminants can be introduced from various sources, includingdetritus shed upstream detectors or preparative systems, such aschromatography columns, or they can be accidentally introduced by directinjection in mircobatch measurements. Even the samples themselves maycontribute to dirtying the cell by forming aggregates with a strongaffinity for the internal optical systems. Once a cell is contaminated,it must be cleaned, either in situ by flushing or more aggressive meanssuch as sonication, as described by Trainoff in U.S. Pat. No. 6,452,672B1 (Issued Sep. 17, 2002), or by removing the cell glass itself andperforming a manual cleaning. While in situ cleaning techniques can beeffective in the short term, most MALS cells must be removed and cleanedmanually on a somewhat regular basis. Flow cell cleaning usuallyrequires some expertise and extreme caution to be certain none of theinner or outer surfaces are soiled by contaminants such as fingerprints,residues and particulates. Further, damage to the cell can occur bothduring manual cleaning, and albeit less frequently, during normalinstrument operation. It is therefore inevitable that the flow cell willneed to be removed from the assembly from time to time.

One limitation of the Phillips cell is the difficulty associated withrealignment of its optical elements after disassembly for cleaning orother maintenance. In the Phillips system, the optical axis is definedaccording to the position of at least three elements, the inlet andoutlet fixtures 103 and 108, each of which house windows through whichthe beam will pass, and the bore 102 of the flow cell 101 itself. If anyof these items are in misalignment, the optical system may fail. What ismore, the position of the connection elements 107 at least partly definethe height of the bore relative to the windows, so any wear or issuesassociated with orientation of the cylindrical connection elements maycause the entire bore to be canted. For these reasons every time thecell is removed from the instrument, every optical element in the chainmust be realigned to ensure the beam is reliably able to pass throughboth windows and the bore of the flow cell without grazing anyinterfaces. Further, the lack of any orienting or registration elementson the glass cell itself contributed to the possibility of misalignmentdue to placing the cell in 180° from its originally aligned position, aswell the possibility of play in the connection elements allowing thebore to be positioned non-parallel to the optical axis defined by theposition of the windows. These limitations made realignment of thePhillips cell cumbersome every time the cell was removed from thesystem.

These problems relating to effectively reproducing the alignmentconditions of the cell relative to the beam when the cell was removedwere addressed by Janik, et. al, in U.S. Pat. No. 5,404,217 (Issued Apr.4, 1995), hereinafter referred to as the '217 patent. Janik describes arigidly connected flow cell manifold housing all elements that may thenbe removed as a single unit from the optical bench of the MALSinstrument. As shown in FIG. 2, the flow cell 201 not contains twoflattened surfaces 202 at the inlet and outlet of the bore. Thesesurfaces serve to register the cell and prevent any rotation thereofrelative to the inlet and outlet manifold halves 203 and 204. Oneembodiment of the Janik invention includes a step 205 milled into theglass cell 201 itself. This step is pressed up against either pins or aseat present in the manifold halves, further ensuring consistentdirectional alignment of the bore with the manifold halves. Eachmanifold half in turn contains an inlet or outlet 206 milled into thetop surface. Fluiding bearing tubing is connected to these ports bymeans of an appropriate fitting. One of these ports 206 directs thesample via a fluid channel to the bore 207, parallel thereto, of theflow cell 201. The flow cell is sandwiched between the manifold halvesand sealed thereto by gaskets or o-rings. Fluid leaving the other end ofthe bore is directed to the outlet port 206 and exists the instrument toproceed to waste, another analysis instrument or a sample recoversystem. Each manifold half also contains an optical window that allows alaser beam to pass along the fluid path in the manifold halves andthrough the flow cell bore. As in the Phillips invention, Janik directsa laser beam through the window within the inlet manifold half 203, andemerging from the window in the outlet manifold half 204 after passingthrough the bore of the flow cell without grazing any surfaces containedtherein. The manifold halves are held together by bolts, and are rigidlyconnected to a base plate 208. Once fully assembled, the flow cellmanifold system is placed within the read head of a MALS instrument towhich the assembly had previously been aligned. This method facilitatesthe reassembly of the instrument after flow cell cleaning and providesreliable reproducibility of alignment for a particular manifold assemblywith a particular flow cell and its associated MALS instrument. ThusJanik enabled a user to disassemble and reassemble the flow cell systemwithout the need to realign the laser after each cleaning.

In all cases discussed thus far, it should be noted that each element ofthe optical system, in particular the flow cell itself, was mated withthe other elements for the life of the alignment. Thus, in all cases,before the present invention it was not possible to replace a flow cellwith a different cell without the need to realign the system. Of coursethe realignment of an optical system adds further complexity to theentire process and is generally performed only by well trainedpersonnel, generally requiring the shipment of the entire system back tothe manufacturer. It is an objective of the present invention to enablethe replacement of one optical flow cell with another within the sameMALS instrument without the need to optically realign the system. It isa further objective of the invention to enable the end user to reliablyreplace the flow cell without specialized equipment or knowhow. It isanother objective of the invention to facilitate the use of distinctflow cells with specific properties, such as refractive indexdifferences or varying bore widths within a MALS instrument. Anotherobjective of the invention is to enable Process Analytic Technology(PAT) to monitor reactions by utilizing bore widths compatible with thistechnology and permitting the replacement of flow cells online withoutthe need to halt the reaction system.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows a flow cell assembly according to Phillips includingfittings for insertion into a light scattering photometer.

FIG. 2 shows elements of the improved manifold and flow cell accordingto Janik.

FIG. 3 illustrates the requirements of degrees of constraint in relationto two flow cell orientations. FIG. 3A shows a sample cell with achannel along the cylindrical axis of the cell.

FIG. 3B shows a sample cell wherein the flow path and the laser path arecollinear.

FIG. 4 shows a side view and a top view of an embodiment of the floatingmanifold of the present invention wherein stacked tolerances arereduced.

FIG. 5 shows top, side and front views of a flow cell compatible with anembodiment of the present invention displaying critical tolerancemeasurements.

FIG. 6 shows side and top views of a floating flow cell manifold whereinalignment elements are in contact with the flow cell in severalembodiments of the invention.

FIG. 7 highlights alignment elements between the manifold and the flowcell utilized in several embodiments of the invention by showing sideand top views of various elements of an inventive assembly. Note the topview does not show read head elements.

FIG. 8 shows side and top views of an inventive embodiment of a verticalflow cell held within a removable, floating manifold and mounted withina read head.

FIG. 9 shows side and top views of a vertical flow cell with criticaltranslational and rotational degrees of freedom restrained as it sitsdirectly within the read head without the need for a separate manifoldelement utilizing the method of exact-constraint design.

FIG. 10 exhibits a side view of a vertical flow cell held within aremovable, floating manifold, and registered directly to the read headwith critical degrees of freedom restrained utilizing the method ofexact-constraint design. The bottom view shows only the flow cell andits registration elements.

FIG. 11 shows noise and baseline levels on the 22° and 90° detectors asthe laser was swept vertically and horizontally parallel to thedirection of the bore of a fused silica flow cell with a bore diameterof 1.2 mm.

FIG. 12 shows the beam position at a cross section of the bore for BSAexperiments used to determine a safe region beam for position variationwhich produce acceptable results.

FIG. 13 shows the variation of two critical alignment measurements on aflow cell according to one embodiment of the invention.

A DETAILED DESCRIPTION OF THE INVENTION

There are many instances in which the flow cell of a MALS or other lightscattering instrument may need to be removed from the instrument inwhich it is housed. In addition to routine maintenance and cleaning, itis sometimes necessary to change the index of refraction of the cell byreplacing it in order to index match the sample solvent with that of theflow cell to improve the signals received at a plurality of angles.Further, while flow cells are generally constructed from some form ofglass, transparent polymers may also serve, under certain conditions, asthe flow cell material, and some solvents are simply incompatible withcertain flow cell materials. Therefore in order improve the versatilityof a single light scattering instrument it is beneficial to enable theend user to replace the flow cell with another under these conditions.Further, as discussed previously, cleaning the flow cell itself can be ameticulous and laborious job requiring significant skill, care and time,and under many laboratory conditions, the light scattering instrument isin almost constant use, and therefore downtime associated with flow cellcleaning can significantly disrupt the laboratory workflow. This isparticularly relevant for PAT applications, wherein the system isflowing constantly as the monitoring of reactions is performed inreal-time, and any significant downtime due to a malfunctioning flowcell can not only disrupt laboratory experiments, but also the veryprocess which is being monitored, and might cause critical stop-reactiontriggers to be missed. Real time monitoring of polymer reactions isdiscussed in depth by J. Y. Gui, et. al., in U.S. Pat. No. 6,635,224 B1(Issued Oct. 21, 2003), incorporated herein by reference, and many ofthe methods discussed therein are also relevant to monitoring of proteinreactions. It is therefore clearly desirous to enable a means wherebylight scattering flow cell can be removed from a light scattering systemwithout the excessive downtime required to realign the systems opticalelements. It is an objective of this invention to provide means toenable a simple “drop in” replacement cell to be used in lightscattering instruments.

As discussed above, the necessity to realign the optical elements in alight scattering system was somewhat mitigated by Janik's '217 patent,by mating a flow cell to a manifold, and providing means including pins,pads and keyed surfaces, retaining, thereby, proper alignment after thecell is removed, cleaned and replaced in the instrument read head, as itwas the complete manifold assembly, not the flow cell itself, that wasinitially aligned with instrument optics. Unfortunately, however, eachelement of the Janik assembly had its own tolerances, and thus it wasnot generally possible to replace a manifold assembly with a secondmanifold assembly and reliably retain optical alignment, as the '217invention stacks at least four levels of tolerances:

-   -   1. The flow cell to the top manifold;    -   2. The assembled top manifold's bottom surface bosses to the        read head;    -   3. The read head to the laser source; and    -   4. The tolerances of the flow cell itself, including the        position of the bore with respect to the glass alignment        surfaces.        Because in the Janik implementation the cell glass was aligned        to the manifold assembly, and then the manifold assembly aligned        to the read head, deviations in exact measurements in each level        of the mechanical interfaces, though each element is within its        required tolerance, could easily result in an instrument where        the originally aligned cell and its associated assembly performs        flawlessly, but a secondary manifold assembly inserted into the        read head resulting in the beam grazing the inner bore, and        producing poor light scattering measurements. It is an objective        of the present invention to improve the interchangeability of        cells and cell assemblies by reducing the stacking of        tolerances.

In order to overcome the limitations of conventional light scatteringcell assemblies, the present invention utilizes a “floating manifold”while maintaining tight controls on measurement specifications of theflow cell itself. In preferred embodiments, the method of“exact-constraint design” (sometimes called “kinematic design,” andthese terms may be used interchangeably in this specification) isemployed to clearly define registration between the measurement cell andthe read head. In our application, exact-constraint design maximizes theefficiency of the restriction the translational and rotational degreesof freedom of the cell within the read head, and in many embodiments isalso employed to restrict the range of motion of optical elementscontained within a floating manifold, including the flow cell. Aschromatographic analysis proceeds to ever smaller sample sizes and theirassociated narrow peaks, it is more important than ever to maintain avery small sample volume required for measurement in any analyticalinstrument, requiring thereby, for most modern applications, a verysmall sample channel cross section. In order to meet these stricttolerance requirements, the present invention minimizes the interfacesover prior art systems while retaining or improving flexibility,robustness, and simplicity the servicing of a light scattering detectionsystem. In particular, many embodiments of the present invention employa floating manifold to reduce the stacking of tolerances and permittingthe flow cell to be independently aligned to both a widow holdingmanifold and the laser containing read head independently. The floatingmanifold assembly and its associated tolerances are thereby effectivelyremoved from the alignment between the beam and the measurement volumewithin the sample channel. Exact-constraint design can be furtheremployed to generalize the invention to many embodiments, configurationsand geometries.

In all cases, the present invention relates to improving lightscattering detection through the illumination of a sample, generallycontained as a liquid suspension, that is contained within a measurementvolume. In order to achieve this end, a light beam, generally a laser,must pass through the measurement volume, and one or more detectors mustbe able to detect light scattered from the illuminating beam by thesample, and therefore, a primary objective of this invention is toenable the simplification of the process of aligning optical elementsand minimizing the number of critical alignment elements, while definingthe maximum allowable tolerance for critical alignment elements. In mostembodiments, including those presented below, the measurement volume isa flow through channel, generally a bore with a circular cross section.It should be noted, however, that the channel need not be circular incross section, but rather, could be of any shape wherein light scatteredfrom the sample can be detected, and while the term “bore” is usedthroughout much of the specification to indicate the path in which thismeasurement volume is contained, in most embodiments and geometries, thebore could still take another form, such as a channel with a square,rectangular, triangular, etc., cross section and still be but oneembodiment of the present invention. In embodiments where the flow pathis co-linear with the beam, transparent windows are a part of theoverall system, whether they are a part of the measurement cell itself,or, as is generally the case, are additional elements held in place by amanifold element such as that used by Janik. Windows may not benecessary in embodiments where the flow is not co-linear with the beamdirection. The primary benefit of the present invention is to provide asystem whereby a beam contained within a read head can reliably beexpected to illuminate a sample volume within a sample cell and enablinglight scattered from the sample volume to be collected by a detector ordetectors also located within the read head, and wherein appropriatelymanufactured sample cell can be swapped into any appropriate read headwithout additional, cumbersome alignment steps. According to thisinvention, this objective is accomplished by registering the flow celldirectly to read head while minimizing extraneous contact points and/orutilizing exact constraint design.

For purposes of illustration and to explore the primary cellorientations many preferred embodiments of the invention, FIG. 3 showsthe constraints needed for two possible flow cell orientations. In bothcases, the flow cells shown 301 and 302 are cylindrical in shape. Thereference axis shown in each example and defined as ground, is attachedto the read head. u_(x) symbols denote translational displacement alongthe x axis. θ_(x) symbols denote rotational displacement along the yaxis. The presence of a bar over the displacement symbols denotes thatthis particular displacement is a degree of constraint, that is,embodiments of the invention with this vertical flow cell orientationrequire these displacements to be constrained, while the absence of abar over the displacement symbols denotes that the displacement is adegree of freedom. In the first example shown in FIG. 3A, the cell 301has what is commonly referred to as a vertical sample channel, that is,the sample channel is a bore drilled along the cylindrical axis of thecell, in this case the y axis is parallel to the bore. This verticalflow cell, wherein the laser crosses perpendicularly to the flow path,requires four degrees of constraint, that is, translationally the cellmust be constrained in the x and z axes, and rotationally about theθ_(x) and θ_(z) axes, however, exact constraint is not required alongthe y axis or rotationally about the θ_(y) axis. Therefore, in order forthe flow cell to be kinematically registered to the read head in whichthe laser is mounted, a minimum of four degrees of constraint arerequired. FIG. 3B shows what is generally called a horizontal (orparallel), cylindrical flow cell, that is one wherein the flow path andthe laser path are collinear, and where the x axis is parallel to theflow cell channel. For this embodiment to be kinmatically registered tothe read head, a minimum of five degrees of constraint are required,that is, translational restraint along the y and z axes and rotationalconstraint in all three θ axes. In order to provide the necessarydegrees of constraint, at least as many distinct contact points orsurfaces as the number of degrees of freedom desired to constrain arerequired. For example on the vertical flow cell shown in FIG. 3A wouldneed at least four contact point of surfaces, thought one mayoverconstrain with 5 or 6 contact points if so desired. It should alsobe noted that there are many engineering choices which can be made tosimplify the number of contact points needed, for example straight linecontact is equivalent to two point contacts. One may choose several waysto implement these contacts via an alignment posts, v-grooves, cones,etc, and some such example embodiments of the invention utilizingseveral methods of exact constraint design are discussed below, all ofwhich are only specific implementations of the invention.

One embodiment of the manifold element utilized in some embodiments ofthe present invention, which is a variation of a traditional flow cellconfiguration is shown in FIG. 4. In a similar manner to the Janikdesign, the optical windows 401 are housed within each section of amanifold element 402 and 403, held into place by retaining washers 404,and sealed by o-rings or gaskets 405. The retaining washers 404 aregenerally threaded to match receiving threaded ports with the manifoldsections. A sample inlet port 406 permits the injection of liquid sampleinto the manifold, and directs the sample to the optical window 401. Thefluid is then directed through a fluid inlet path 408. The flow cell 410is sandwiched between the two manifold sections 402 and 403 and the endsof the cell bore 411 are sealed between the fluid inlet and outlet paths(408 and 409 respectively) by o-rings or gaskets 412. The x-y plane isdefined by three precision milled posts 415 upon which the bottom planarsurface of the cell is placed. The posts may have a flat top surface incontact with the bottom surface of the cell, or they may behemispherical, in which case apex of each hemisphere makes contact withthe planar surface at only one point, and thus exactly defining theplane upon which the cell rests within the manifold. An incorporatedstep 416 within the flow cell 410 is placed in contact with twoalignment pins 417, generally precision milled cylinders. The planedefined by the step 416 when registered against the single vertical lineof contact of each post 417 defines the cell location in the horizontaldirection, permitting thereby alignment of the bore 411 with the fluidpathways 408 and 409 and the optical windows 404. The two manifoldsections 402 and 403 are held together by bolts 414 placed through onemanifold section and into an engaging threaded hole in the othermanifold section. In contrast to the Janik assembly, the manifoldsections are connected beneath the flow cell rather than above it.Another critical element of the present inventive flow cell assembly arethe three vertical holes 412 machined into the bottom surface of themanifold, which allow aligning elements of the read head direct accessto the bottom surface of the cell as discussed below. In accordance withexact-constraint design, the three alignment posts in contact with theplanar bottom surface of the cell and the two alignment pins in contactwith the step define the relative position of all elements of themanifold to all elements of the flow cell.

The fully assembled manifold structure can then be placed in analignment fixture to verify that the optical windows 401 and bore 411are properly aligned, such that the laser beam passes through thewindows and bore without intersecting any surfaces that might causeimproper light scattering measurements. Once this alignment is verified,the cell assembly may be inserted into the read head.

Let us now take a closer look at the flow cell utilized in this exampleembodiment of the invention. The reduction of stacked tolerances in thepresent invention and strict tolerance requirements for key elements ofthe flow cell manufacture permit independent glass cells to beinterchanged with independent light scattering instruments. The cell 501in FIG. 5 has a right cylindrical shape with the bore 502 machinedthrough a radius of the cylinder. Two flat faces 503 are machined intothe cell at the intersection of the bore 502 with the circumference ofthe cell 501. A flat step 504 is machined into one edge of the cell,generally parallel to the bore, however this preferred orientationshould not be considered limiting. In this embodiment, the most criticalmeasurements of manufacture are labeled d₁ and d₂ in FIG. 4. Thevertical distance d₁ between the edge of the bore 501 on each of theflat faces 503 and the flat bottom of the cell is one such criticalmeasurement. The second, d₂, is the horizontal measurement between theedge of the bore 502 and the inner edge 505 of the step 504. It is alsocritical that the bottom surface of the cell 506 be flat across theentire surface within strict tolerances. When these critical elementsare fabricated within the strict tolerances required, problemstraditionally associated with alignment of replacement cells will not bepresent with the inventive assembly presently disclosed.

The next element of the inventive design is the read head which isdesigned to mechanically register directly to the flow cell rather thanto the manifold as in previous MALS system designs. FIG. 6 shows somecritical alignment elements of the cell/read head interface for anembodiment shown in FIG. 4. As mentioned above, the manifold assembly601 holding the flow cell 602 floats within the read head 603 withoutnecessarily making rigid contact thereto. The three holes 604 machinedinto the bottom of the manifold assembly allow vertical alignment posts605 that are rigidly connected to the bottom of the read head to comeinto direct contact with the flow cell glass, defining the horizontalplane of the flow cell irrespective of any lose tolerances between thefloating manifold and cell. As with the flow cell bottom plane definingcontacts within the manifold, the vertical alignment posts of the readhead may be either precision machined flat topped posts, generallycylindrical in body shape, though they may take other forms as well, orthese posts may have hemispherical ends, such that a single point ofcontact for each of the three posts defines the plane upon which thecell sits within the read head. Bosses 606, which are generally roundedprotuberances from the body of the read head, provide horizontalalignment by coming into contact with the precision machined verticalsurface of the flow cell step 607. One ball plunger 608, generallyintegrated into the read head, applies a horizontal force to the cell,pushing the cell step against the rounded bosses 606 ensuring horizontalalignment of the cell bore to the read head mounted laser. Horizontalalignment is guaranteed by the strict tolerance d₂ discussed above. Asecond ball plunger 609 may be employed to provide additional pressuredriving the cell step 607 against the alignment bosses 606 andadditionally improve the stability of the seating of the floatingmanifold 601 within the read head 603. To improve clarity in FIG. 6 thehorizontal planar alignment posts between the bottom surface of the celland the manifold (elements 415 of FIG. 4) are not shown, however thepins 612 defining horizontal alignment (elements 417 of FIG. 4) betweenthe manifold and the cell step 607 are shown to facilitate thevisualization of the independent horizontal alignment of the cell 602 tothe read head 603 as well as the cell 602 to the floating manifold 601.Once the manifold assembly is seated within the read head a compressibleelement 610, such as a wave washer, gasket or o-ring is sandwichedbetween the flow cell 602 and a top plate 611 which is connecteddirectly to the read head 603 without coming into contact with themanifold assembly 601, thus maintaining the floating nature of themanifold while simultaneously applying a downward force, pushing theflow cell 602 onto the alignment posts 605 and defining, thereby thehorizontal plane of the bottom of the cell. Thus vertical alignment ofthe cell is likewise guaranteed by the strict tolerance d₁ discussedabove. Therefore, a laser mounted in the read head that is aligned toany appropriately fabricated flow cell adhering to the criticalspecification tolerances d₁ and d₂, will be properly aligned to anyother flow cell contained in any other floating manifold which alsoadheres to the d₁ and d₂ specifications, and it is by this inventivemeans of decreasing the stacked tolerances and controlling a handful ofstrict specifications, that interchangeable flow cells between lightscattering detectors is enabled. As was the case with the alignmentelements between the cell and the manifold, exact-constraint design isemployed by the three alignment posts 605 in contact with the planarbottom surface of the cell and the two alignment bosses 606 in contactwith the step define the relative position of all elements of themanifold to all elements of the flow cell. This configuration of flowco-linear with the beam is sometimes referred to as a horizontal flowcell. In this embodiment the critical degrees of freedom that must beconstrained are pitch, yaw, roll and translation perpendicular to thebeam, however, the position of the cell parallel to the beam is not ascritical to an effective light scattering measurement, as smalldeviations will vary only the position along the bore of the scatteringvolume, but not cause issues relating to the beam actually passingthrough the sample volume which would be the case if, for example, theposition of the cell were shifted perpendicular to the beam, or out ofthe X-Y plane by the measurement of the bore diameter. Therefore we willconsider that this embodiment requires five degrees of freedom to beconstrained.

In order to further enhance the reproducibility and maintenance ofalignment of interchangeable flow cells and cell assemblies disclosedthus far, a preferred embodiment of the invention shown in FIG. 7employs means for applying a force between the manifold and the flowcell to maintain proper alignment between the windows 701 and the cellbore 702. In this embodiment a top manifold plate 703 is rigidlyconnected to the main manifold body elements 704. The top manifold plate703 is generally connected to the manifold body elements 704 by means ofthreaded screws (not shown), but may also be connected by other meanssuch as a clamping mechanism. A vertical force providing means 705, suchas a wave washer, spring, gasket or o-ring, provides a downward forceF_(v) (indicated in FIG. 7 by arrows) on the flow cell 706, activelypushing it against the manifold alignment posts 707, which define theplane upon which the bottom of the flow cell 706 rests. As these forceapplying elements assure the bore 702 is in alignment with the windows701, it guarantees that the beam 708 generating laser housed in the readhead 709 will pass properly through both the windows 701 and the bore702. It is important to note that the cell-to-read head verticalalignment posts 605 shown in FIG. 6 are still present in thisembodiment, though they are not shown for the sake of clarity in FIG. 7.And as shown in FIG. 6, a read head top plate 710, though modified asshown in this figure, still enables the application of a downward forcedirectly to the flow cell, driving the cell against the read headalignment posts (not shown). In the currently presented embodiment, theread head top plate itself acts as a spring element pushing downward onthe flow cell without the need of a wave washer, o-ring or other extraforce generating element. This is enabled by the read head top plate 710having a slight concave curvature, and thus when rigidly connected tothe top of the read head 709 it deforms slightly, with the resultingmechanical stress providing the downward pressure to the flow cell 706.The portion of the read head top plate in contact with the top of theflow cell 706 extends through a hole in manifold top plate 703. The topview presented in FIG. 7 does not show the elements of the read head inorder to clearly show the critical elements of the presented embodiment.In order to provide a horizontal force F_(h) driving the flow cell 706and its incorporated horizontal alignment step 711 against the manifoldhorizontal alignment pins 712, a spring element 713 is connected byconnection means such as threaded screws or bolts 714 screwed into eachmanifold element 704. This spring element, which may be a properlyformed strip of sheet metal, or leaf spring, the deformation of which,when connected to the manifold and in contact with the flow cellprovides the desired horizontal force. It should also be noted, that inthis embodiment, the spring element 713 in contact with the flow cell704 may also act as the contact point for one or more of the forcegenerating ball plungers 508 and 509 that apply force to the flow cellto drive it against the read head rounded horizontal alignment bosses506, the spring element 613 can therefore both apply the necessarycell/manifold horizontal alignment force as well as providing protectionto the cell itself which might otherwise be scratched by the contact ofthe ball plunger 608 or ball plungers 608 and 609 if they were to makedirect contact with the cell surface when the manifold/cell assembly isplaced into the read head. It should also be noted that the springelement 713 need not be, and generally is not, the same height as theflow cell 704. In general the spring element extends only partially upthe up the height of the cell, thus permitting optical access in ahorizontal plane, to the flow cell bore 702, permitting, thereby, lightscattering detectors to be placed at any number of angles about the boreon both sides of the cell. Alternately, the spring element may be a leafspring with an opening along its horizontal axis, permitting opticalaccess in a horizontal plane to the bore 702 while applying the desiredhorizontal force near the top and bottom of the height of the cell, thusproviding a more uniform pressure. This embodiment not only promotessimple alignment of the elements during assembly, but mitigatesmisalignment during operation due to such causes as vibration or jarringof the instrument by actively pushing the elements back against theiralignment fixtures.

It should be noted that the right cylindrical shape common to many lightscattering detectors, such as the DAWN HELEOS (Wyatt Technology, GoletaCalif.), should not be considered a limitation of the present invention.For example, the lensed flow cell described by Trainoff in U.S. Pat. No.982,875 (issued Jul. 19, 2011) could also be compatible with the presentinvention. In addition perpendicular flow cells discussed previouslycould also benefit from the present invention, extending the invention'sutility to use with modern UHPLC systems. Further, while this disclosureis primarily concerned with MALS systems, use of the term MALS shouldnot be considered limiting, as the invention disclosed herein is alsocompatible and beneficial to other flow through light scatteringinstruments including low angle light scattering (LALS) and right anglelight scattering (RALS) instruments. In addition, dynamic lightscattering (DLS) instruments also benefit from the present invention.DLS measurements can be taken independently or concurrently with MALS,LALS or RALS data. All light scattering instruments involving flow orstop-flow instruments are compatible with the present invention.Therefore throughout the specification the term MALS is generally used,as is the parallel flow cylindrical cell merely to simplify explanation,and thus should not be considered limiting at any point nor should anyembodiment discussed herein as an exemplar be considered limiting.

For another example of how exact-constraint design is used to simplifythe mounting and alignment of the sample cell according to the presentinvention, consider the embodiment including a “vertical” flow cellaccording to the invention shown in FIG. 8. As discussed above, thevertical flow cell 801 is one wherein the flow is perpendicular to thedirection of the beam 812, that is to say the beam crosses the samplelaterally with respect to the direction of the sample flow. In this casethere are fewer critical degrees of freedom in need of constraintassociated with the positioning of the cell. With vertical flow throughthe cell and horizontal laser beam perpendicular to the flow, it iscritical to constrain the position of the cell in the x-y plane. Pitchand yaw constraints are also important in order to properly align thedetectors to the measurement volume near the center of the cell.However, roll, or rotation about the center of the cell, and position inthe z direction are less critical, and therefore these degrees offreedom need not be constrained exactly by means of exact-constraintdesign for embodiments that use a vertical flow cell. There are variousmeans by which the cell may be positioned within the read head, and, asthe containing manifold need not house optical windows, the registrationof the cell to the read head is made yet simpler. FIG. 8 shows anembodiment of the invention with a vertical flow cell 801 which utilizesa floating manifold similar to that described in above horizontal orco-linear flow embodiments. The flow cell 801, as before, is sandwichedbetween a top and bottom manifold 802 and 803. In this case the manifoldserves primarily to provide inlet 804 and outlet 805 means to themeasurement channel 806. A length of tube 807 is seated in the inletport 804 with a fitting 808. Similarly the outlet tubing 810 is fittedinto the outlet port 805 with a fitting 80.9 The manifold may houseother elements such as inline filters, flow distributors, delay volumes,and sealing means, such as o-rings, etc., in its body, and/or within theflow path between the inlet and/or outlet tubing and the measurementchannel 806. Holes 811 machined into the manifold 802 permit access ofthe laser beam 812 to the measurement channel 806. Alternately, theholes 811 may give optical access to the measurement channel to lightscattering or other detectors, and the laser may be directed ratherthrough the open side of the manifold, for example, perpendicular to theopenings 811. It should be noted that in this configuration, the holesneed not contain optical windows, and maybe significantly oversized,thereby obviating the need to carefully align laser or detector accessthere through. However, windows or other optical elements such asfilters may be placed into these portals 811 as appropriate. The flowcell 801 may incorporate an alignment step 812. Further, either the topor bottom manifold 802, 803 has machined, there trough means, such as ahole or holes 813 by which alignment posts 814 extending from the readhead may make contact with the planar bottom surface of the cell,defining thereby the x-y plane of the cell 801 relative to the readhead. Alignment bosses 815 incorporated into the read head come intocontact with the step, defining thereby the position of the measurementchannel 806 along the y-axis relative to the laser beam. In most casesthe position of the channel along the x-axis should also be constrained,particularly when the measurement channel has a circular cross section.In this case the alignment step 812 may be L shaped, and a third,alignment boss 816 will come into contact with the alignment step alongthe y-axis 817. When the all three alignment bosses are in contact withthe step, the position of the measurement channel 806 will be exactlyconstrained in the x-y plane. Force may be applied from the read head tothe cell from the side to drive the step onto the alignment bosses bymeans such as a ball plunger 818. And force may be applied opposite thealignment posts 814 within the read head 811 to the cell 801 or to themanifold 802, by means such as a wave washer 819, o-ring or gasket, todrive the cell 801 directly against the alignment posts 814. Thus, onceagain, the manifold floats, and it is the cell is directly registered tothe read head. However, in this embodiment, it is not necessary thatthere be optical alignment between any elements within the manifold tothe cell, thereby simplifying the step of combining the cell-manifoldassembly.

Further simplifications are made to the system by more efficientutilization of exact constraint design, and permitting the manifoldelements to float more completely or to be themselves incorporated intothe read head. Consider the example embodiment shown in FIG. 9 where theoutlet port 901 is an element of the read head itself, and the read head902 is shaped so as to take advantage of the cylindrical nature of thesample cell 903. The V shaped end 904 the read head cavity comprises twovertical flat faces 905 and 906. When the cell 903 is placed into thecavity within the read head 902, the two lines of contact between thefaces of the V channel 904 and the cylindrically shaped flow cell 903constrain the cell 903 in all critical degrees of freedom. A horizontalforce is applied to the cell by means such as one or more ball plungers905 to firmly drive the outer cell wall against the V surfaces. Thesample outlet 901 from the channel 906 is sealed with an o-ring 907 orgasket to with the outlet port 901 incorporated into the read head 902.The inlet into the measurement channel 906 is coupled with a sealingmeans 908, to an inlet port assembly 909; this assembly applies adownward pressure onto the cell, causing the o-rings 907 and 908 toseal. This pressure can be applied by any number of means such as a topflow assembly 909 comprising a flow port 910 to which inlet tubing 911may be connected by means of a fitting 912, and which is screwed into atop plate 913 connected to the read head 902 itself by means such asscrews 914, bolts or clamps. In this embodiment, the manifold is almostcompletely removed from the system. Within in the V section of the readhead either the laser 915 or a photodetector may be placed. If the laseroccupies this space, it may be beneficial to employ more than one forcegenerating elements 905 on the opposite side of the cell in order topermit optical access to the beam 916 as it emerges from the far side ofthe cell. For example, by placing two ball plungers 905 each 45° fromthe beam path, equal pressure can easily be applied to drive the cellinto the V channel. If the cell is of sufficient height, two ballplungers could also be used in line with the beam 916, but placed aboveand below its eventual intersection with the read head, thus applyinguniform force directly into the V channel while still being out of thepath of the laser and permitting the emerging beam to fall upon a lasermonitor element 917. Additionally other means of applying horizontalpressure may be utilized, such as a leaf spring containing a hole toallow passage of the beam through the cell. While it may be simplest toinclude the laser 915 at the apex the V channel 904 of the read head902, there may be many reasons to choose another configuration, such asthe beam 916 traversing the measurement volume perpendicular to thedirection of the V channel 904, or for that matter, it may beadvantageous for the beam 916 to traverse the cell 903 at any otherarbitrary angle in order to maximize both the uniformity of forcedriving the cell 903 against the V groove 904 while also maximizing theefficiency of the positioning of light scattering detectors 918 relativeto the beam direction.

FIG. 10 illustrates elements of a similar embodiment also employingexact-constraint design. In this case the “three-V” constraint system isused. By incorporating three precision machined V grooves 1001 into thecell 1002 itself, and incorporating three precision positioned spheresor, perhaps more practically, posts 1003 with hemispherical tops 1004,the cell 1002 can have all critical degrees of freedom constrained withno further alignment necessary. This embodiment can be utilized eitherwith the outlet port incorporated directly into the read head itself (asin the example shown in FIG. 9), or the inlet and outlet flow to thecell 1002 can be restricted with a floating manifold unit as shown inFIG. 9. In this example the manifold comprises top 1005 and bottom 1006portions connected by bolts 1007, and each manifold element comprises aport element 1008, with sealing means 1009 located between each elementand the cell itself 1002. Additionally, the floating manifold must allowaccess to the three hemisphere topped posts 1003 to the V grooves 1001incorporated into the cell 1002 by means such as one or more holes 1010contained within the bottom manifold element 1006. The port elements1008 may also hold additional elements such as inline filters, flowdistributors, and/or dead volumes. It should be noted that the floatingmanifold presented may also replace the read head incorporated outletport in other embodiments, in particular that shown in FIG. 9. Again, asthe positioning in the z-axis is not critical some loosening of thetolerances in any vertical positioning elements is permitted withoutloss of signal quality. It should be noted that the incorporation ofadditional geometries into light scattering cells, such as the V groovesmachined into their surface in the embodiment presented in FIG. 10, maygive rise to unwanted reflections and stray light issues under certaincircumstances, and for that reason it is often desirable to make thescattering cells of the simplest geometry possible, such as thetraditional cylinder with a bore drilled through a diameter. Ifreflections and stray light are of particular concern, this embodimentcan employ a section of black, or appropriate wavelength absorbing,glass adhered or contact bonded to the main body of the cell, where thisregion of black glass is the area containing the V grooves, or othernecessary exact constraint design geometries, hence decreasing thelikelihood that these added geometries will cause optical problems.

In order to investigate the efficacy of the inventive apparatus withregard to the interchangeability of flow cells and/or manifolds for agiven light scattering detector, experiments were performed using aretrofitted DAWN® HELEOS® II light scattering photometer (WyattTechnology Corporation, Goleta, Calif.). These experiments furtherhelped to define the tolerances on the key specifications which must bemet in order for the system to perform properly. The flow cells used inthese experiments were fabricated from fused silica and had dimensionsof approximately 30 mm in diameter, 11 mm in height with a bore diameterof 1.2 mm in the same general configuration as the cell shown in FIG. 4.A control cell was mounted in the floating manifold and placed in theretrofitted read head of the light scattering instrument. The laser wasthen aligned to the control cell such that it passed through the centerof the bore of the flow cell. The laser was a 658 nm diode laser withcorrective optics designed to minimize the beam width common in MALSdetectors.

Once properly aligned the laser was swept horizontally and verticallywhile the 90° and 22° light scattering detector signals were monitored.This test determined a bore diameter safe region by measuring the laserposition offset from the center of the bore that yielded results atacceptable baseline and noise levels. Holding the laser fixed in onedirection and sweeping it both vertically and horizontally from 350 μmfrom the center of the bore to −350 μm had no appreciable effect onnoise levels and only affected baseline levels in the low angle detectoras shown in FIG. 11.

The next experiment was to perform standard aqueous chromatographyinjections of bovine serum albumin (BSA) with the laser located atpositions offset from the center of the bore at radii of 340 μm and amore restrictive 170 μm. The positions of the laser in a cross sectionof the bore are shown in FIG. 12. The resulting weight averaged molarmasses were calculated for each run are displayed in Table 1. Theacceptable range for BSA molar mass is 63.08 kDa-69.72 kDa. Therefore,as the results of Table 1 show, only at two positions did themeasurements fall outside the specification range.

TABLE 1 BSA weight averaged molar mass measurements with laser atdifferent distances and positions from the cross-sectional center of thecell bore BSA Measured Molar Mass (kDa) Position r₁ (170 μm) r₂ (340 μm)A 63.52 62.66 B 67.10 68.59 C 66.55 67.31 D 64.52 62.57

The results shown in the above experiment defined a probable safe regionwithin 170 μm radius about the center of the bore cross section. Theseresults were confirmed by fractionating 200 kDa and 30 kDa polystyreneparticle suspensions with a chromatography system using toluene as themobile phase. The acceptable molar mass range for 200 kDa polystyrene is190-210 kDa, and 25-35 kDa for the 30 kDa polystyrene. The results ofthese runs are shown in Table 2. Not only did every run fall well withinthe specification, the results were very consistent, thus confirming thesafe region of at least 170 μm radius about the cross sectional centerof the bore.

TABLE 2 Polystyrene latex injections into a size exclusionchromatography system with toluene as a mobile phase. Measured MolarMasses (kDa)-r = 170 μm Position 200 kDa 30 kDa O 205.3 29.36 A 204.629.22 B 203.8 29.27 C 204.4 29.22 D 204.6 29.31

The results of the above tests indicating a safe region of 170 μm radiusinside the bore suggested that aligning to the read head with thisinventive design provides adequate accuracy in bore position to be ableto interchange flow cells without re-aligning the laser. The deliverablespecification for bore position tolerance is +/−75 μm with respect tothe alignment features previously discussed as measurements d₁ and d₂,thus even maximum deviation within the specification still falls wellwithin the safe region. The measured distances of d₁ and d₂ for a seriesof ten test cells and recorded historical measurements of cells arepresented in FIG. 13. Historical data is denoted by the small diamondswhereas the larger squares indicate test cells. As noted in FIG. 13, thelargest deviation between test cells in d₂ is 84 μm, and the largestdeviation in d₁ is 52 μm, well within the safe region discussed above.In order to practically confirm the efficacy of the invention, thecontrol cell discussed above was replaced within the read head of theretrofitted MALS instrument with each of the test cells indicated assquare markers in FIG. 13. After interchanging cells, with no alignmentprocedure performed, BSA and polystyrene injections were made, asbefore. As is evident from the data presented in Table 3, all of thecells provided data well within the acceptable values, thus confirmingthe ability of interchange flow cells with the inventive systemdescribed herein.

TABLE 3 Chromatography results for ten test cells replaced within aretrofitted read head and utilizing a floating manifold head withoutperforming realignment procedures.. 1 (control) 2 3 4 5 6 7 8 9 10 200kPS 202.3 203.8 203.7 203.5 203.9 203.3 203.5 203.4 203.6 202.4 MM 30k PS29.11 29.12 29.2 29.1 29.15 29.12 29.18 29.14 29.06 29.12 MM BSA MM63.83 64.71 63.5 65.36 65.35 65.13 64.2 65.12 66.18 65.86

While the benefits of the present invention are useful to lightscattering detectors in general, they are of particular relevance in thefield of Process Analytic Technology (PAT) where ongoing reactions aremonitored for significant changes to indicate when a particular reactionis complete, or the product being produced has reached the limits of itsacceptable range. For example, in the polymer industry PAT is used tomonitor reactions which produce polymers from monomeric species. See,for example, U.S. Pat. No. 9,568,462 B2, “Methods and instrumentationfor during-synthesis monitoring of polymer functional evolution,” byW.F. Reed (Issued Feb. 14, 2017). As Reed explains, in continuousreaction processes, where one product composition is changing toanother, it is essential to be able to monitor the online state of theproduct and determine the point of acceptable changeover of one productcomposition to another. Reed describes monitoring molar mass values inorder to determine the amount of the fundamental residual monomer stillpresent in the reaction stream while producing the desired polymer.

Another example of an industrial process that can benefit from PATincludes the purification of a given product. This process involvespurification steps wherein a product containing contaminants is passedthrough a system intended to permit passage of the product but retainthe majority of contaminants. For such systems the purificationmechanism often has a limit to the quantity of contaminants which may beretained. Once this practical threshold is reached, contaminants maybegin to “break through” the purification device and become part of theproduct stream. One example of this type of process frequently used inthe pharmaceutical industry is flow-through process chromatography, andvariations include flow through hydrophobic interaction chromatographyand flow through ion exchange chromatography. For these cases proteinmonomer, dimer, and higher order oligomers are all contained within anunpurified material. Only one of these species is desired in the finalproduct, e.g. the protein monomer. The chromatographic system is tunedsuch that the desired species, e.g. monomer, passes through thechromatographic system, and the undesirable species, e.g. dimers andhigher oligomers, adhere to surfaces within the chromatographic system.When material first begins to flow through the system, there is a largesurface area available to which contaminants can adhere. Eventually, theadhesion sites become filled, and more and more undesirable species passthrough the chromatographic system. This product stream elution maybemonitored by a system, such as one comprising light scattering andconcentrations detectors. This detection system can determine thereal-time value of the molar mass, which increases as more of the eluentcontains oligomers rather than almost exclusively monomer species. Anincrease in the monitored value of the molar mass beyond a pre-set giventhreshold marks the end of the purification run. Another PAT methodinvolves affinity chromatography, also known as bind and elutechromatography. In this case, the desired sample is passed through ahighly selective column specifically chosen such that the desiredmolecule interacts with the stationary phase and is thus bound withinthe column and is thus separated from the stream. Thus the contaminants,rather than being retained, are flushed first and the desired moleculesare retained until a change is made to the solvent flow which releasesthe retained, purified sample. Similarly to, flow-through processchromatography, the molar mass of the eluting fractions can be monitoredfor changes which indicate the end of the purification process andtrigger the change in mobile phase required to release the retained,purified sample from the column, which is then collected.

Utilizing the present invention in a PAT system offers the advantage ofbeing able to not only replace the flow cell quickly, but, moreimportantly, to permit the replacement of key elements of the systemreliably without the need to replace an entire instrument within theprocess environment. With the embodiments of the present invention usedwithin a PAT system, the flow cell can be reliably removed and replacedwithout the need to requalify a new instrument in the process stream,and thus, for the first time, a process engineer without extensiveexperience with optical instrumentation, can, with little downtime,perform simple, turn-key maintenance to a PAT-MALS system. Whereaspreviously a dirty or permanently fouled cell might require an opticalexpert and/or extensive training to clean, and a permanently fouled cellmay require the replacement of a complete instrument, and in all casesreplacing the cell would require extensive re-evaluation andre-qualification within the PAT system, the present invention makes MALSinstruments practical within a PAT environment.

Further, while traditional chromatography systems generally utilizeextremely dilute solutions for measurement of properties of the liquidsamples separated therewith, some PAT environments involve a much widerrange of sample concentration. Some embodiments of the present inventionenable the use of a light scattering detectors with very littlepreparative work and a much wider variation in sample composition thanis true with traditional chromatography measurements. As discussedabove, the critical alignment measurements d₁ and d₂ when combined withknowledge of the cross sectional radius of the sample bore define a“safe region” where variation in the position of the beam still producesquality results. Therefore, in PAT systems, one may increase the radiusof the cross section of the bore two, three, four or more times whilestill mitigating beam steering due to changes in sample compositionthroughout the reaction process. Because of the decreased tolerancestacking in the present invention, utilizing these wide bore samplecells is possible in PAT systems.

As will be evident to those skilled in the arts light scattering andmacromolecular characterization, there are many obvious variations ofthe methods and devices of our invention that do not depart from thefundamental elements that we have listed for their practice; all suchvariations are but obvious implementations of the invention describedhereinbefore and are included by reference to our claims, which follow.

What is claimed is:
 1. An optical flow cell assembly comprising: atransparent flow cell; a manifold configured to contain the flow cell; aread head comprising a beam generating light source aligned to emit abeam of light along an axis of the read head; and wherein the read headis configured to be directly registered to the flow cell, therebyaligning the flow cell to the read head, wherein the read head furthercomprises a plurality of read head vertical alignment posts configuredto contact at least one planar alignment surface of the flow cell todefine a vertical read head-to-flow cell alignment plane perpendicularto the axis, a read head top plate configured to drive the at least oneplanar alignment surface against the read head vertical alignment posts,at least one read head horizontal alignment first surface bossconfigured to contact a first surface of an alignment step of the flowcell to define a horizontal read head-to-flow cell alignment planeperpendicular to the vertical read head-to-flow cell alignment plane, atleast one read head horizontal alignment second surface boss configuredto contact a second surface of the alignment step of the flow cell,wherein the second surface is perpendicular to the first surface, and aball plunger configured to drive the alignment step into contact withthe at least one read head horizontal alignment first surface boss andthe at least one read head horizontal alignment second surface boss,wherein the plurality of read head vertical alignment posts is distinctfrom the read head top plate, is distinct from the at least one readhead horizontal alignment first surface boss, is distinct from the atleast one read head horizontal alignment second surface boss, and isdistinct from the ball plunger, wherein the read head top plate isdistinct from the at least one read head horizontal alignment firstsurface boss, is distinct from the at least one read head horizontalalignment second surface boss, and is distinct from the ball plunger,and wherein the at least one read head horizontal alignment firstsurface boss is distinct from the ball plunger, and wherein the at leastone read head horizontal alignment second surface boss is distinct fromthe ball plunger.
 2. The optical flow cell assembly of claim 1 whereinthe manifold comprises: a liquid sample inlet port; a liquid sampleoutlet port; an inlet path coupled to an outlet of the inlet port; anoutlet path coupled to the inlet of the outlet port; and wherein theinlet path and the outlet path are configured to direct a flow of aliquid sample from the inlet port through the flow cell and out of theoutlet port.
 3. The optical flow cell assembly of claim 2 wherein themanifold further comprises vertical holes configured to allow access tothe at least one planar alignment surface.
 4. The optical flow cellassembly of claim 1 further comprising a compressible element configuredto generate a vertical force between the flow cell and the read head todrive the at least one planar alignment surface against the plurality ofread head vertical alignment posts.
 5. The optical flow cell assembly ofclaim 4 wherein the compressible element comprises a wave washerconfigured to be placed between the flow cell and the read head topplate configured to be rigidly connected to the read head.
 6. Theoptical flow cell assembly of claim 1 further comprising a springelement configured to generate a horizontal force between the manifoldand the flow cell to drive the flow cell against the plurality of readhead vertical alignment posts.
 7. The optical flow cell assembly ofclaim 6 wherein the spring element comprises a leaf spring configured tobe rigidly connected to the manifold and configured to be in contactwith the flow cell.
 8. The optical flow cell assembly of claim 7 whereinthe leaf spring defines an opening along a horizontal axis of the leafspring, wherein the opening is configured to permit access to the flowcell.
 9. A method comprising: containing an optical flow cell within amanifold such that the optical flow cell is aligned to the manifold; andregistering directly the flow cell to a read head with read head-to-flowcell alignment elements, thereby aligning the flow cell to the readhead, wherein the read head comprises a beam generating light sourcealigned to emit a beam of light along an axis of the read head, whereinthe read head further comprises a plurality of read head verticalalignment posts configured to contact at least one planar alignmentsurface of the flow cell to define a vertical read head-to-flow cellalignment plane perpendicular to the axis, a read head top plateconfigured to drive the at least one planar alignment surface againstthe read head vertical alignment posts, at least one read headhorizontal alignment first surface boss configured to contact a firstsurface of an alignment step of the flow cell to define a horizontalread head-to-flow cell alignment plane perpendicular to the verticalread head-to-flow cell alignment plane, at least one read headhorizontal alignment second surface boss configured to contact a secondsurface of the alignment step of the flow cell, wherein the secondsurface is perpendicular to the first surface, and a ball plungerconfigured to drive the alignment step into contact with the at leastone read head horizontal alignment first surface boss and the at leastone read head horizontal alignment second surface boss, wherein theplurality of read head vertical alignment posts is distinct from theread head top plate, is distinct from the at least one read headhorizontal alignment first surface boss, is distinct from the at leastone read head horizontal alignment second surface boss, and is distinctfrom the ball plunger, wherein the read head top plate is distinct fromthe at least one read head horizontal alignment first surface boss, isdistinct from the at least one read head horizontal alignment secondsurface boss, and is distinct from the ball plunger, and wherein the atleast one read head horizontal alignment first surface boss is distinctfrom the ball plunger, and wherein the at least one read head horizontalalignment second surface boss is distinct from the ball plunger.